February 2019

Customer Data: Use of Seraseq ctDNA Reference Samples to Validate Detection of Low Frequency Variants in a cfDNA-based NGS Lung Cancer Panel

At the recently-concluded 2018 AMP Meeting, researchers at the New York Presbyterian Hospital (NYPH) and Weill Cornell Medical Center (WCMC) presented a poster1 on the validation of an Oncomine™ cell-free DNA Lung Assay using ctDNA NGS standards developed by SeraCare (Seraseq® ctDNA v2 Reference Materials),2 for detecting low frequency somatic mutations at a range of DNA inputs to stress-test the NGS assay sensitivity.

Table 1: List of cfDNA gene variants in the Seraseq ctDNA v2 reference samples2 concordant with Oncomine™ cfDNA Lung Cancer Assay.1

In the study, low frequency variants (11 – see Table 1) in six lung cancer genes - EGFR, KRAS, BRAF, TP53, PIK3CA, and ERBB2 – covered in the Oncomine cfDNA Lung Cancer Assay were evaluated using plasma-based Seraseq ctDNA reference standards at AF0.1%0.5%1%, and 2%, and DNA inputs of 1ng, 5ng, 10ng, and 20ng. Testing was performed in triplicate. Sequencing on the Ion S5 platform yielded a mean coverage range between ~27,000x (1 ng) to ~96,000x (20 ng). The molecular coverage ranged between ~230x (1ng) to 2,855x (20ng), allowing accurate detection of low-frequency variants and reliable determination of limit of detection (LOD) cutoff values.  

Figure 1 shows the correlation between expected and observed VAFs at 1 ng and 20 ng cfDNA inputs, suggesting that the assay is robust enough to call VAFs at low DNA inputs. However, the study found high CVs for VAFs at the lowest input of 1 ng (CV range of 44% to 138%) compared to a range of 18-51 at 10 or 20 ng DNA inputs.

The study concludes that the Oncomine cfDNA Lung Assay, as validated with plasma-based contrived reference samples, is a sensitive cell-free DNA-based NGS panel for reliable and reproducible detection of specific low-frequency variants in cancer plasma samples. Careful validation and determination of LOD cutoff values in the context of DNA input is warranted.  

Figure 1: Performance of Oncomine™ Lung cfDNA Assay was measured across four VAF levels (0.1%, 0.5%, 1%, and 2%) of 11 targeted gene variants by input DNA. Graphical representation of the regression analysis for (A) 1 ng and (B) 20 ng input cfDNA is shown. There was an excellent concordance between the expected and observed VAFs using the quantitative multiplex reference standard. The correlation coefficients (R2) values were 0.90 and 0.99 for 1 ng and 20 ng input cfDNA, respectively.1

For more details about SeraCare’s ctDNA reference samples please visit our liquid biopsy portfolio.

This blog was authored by Omo Clemente, PhD.

1. Lin Cong, Jordan Baum, Phyllis Ruggiero, John Sipley and Hanna Rennert, Department of Pathology, New York-Presbyterian Hospital and Weill Cornell Medicine, New York, NY; "Assessment of the Oncomine™ cell-free DNA Lung Assay to Detect Low-Frequency Mutations using Reference Material," 2018 AMP Poster presentation (poster #: TT058), San Antonio, TX, Nov 1-3, 2018.
2. Seraseq ctDNA v2 Reference Materials, manufactured by SeraCare Life Sciences Inc., 37 Birch Street, Milford, MA 01757.